This application addresses broad Challenge Area (08) Genomics and specific Challenge Topic, 08-ES-104: Identification of alterations in epigenetic marks related to environmental exposures. In the US, exposure to pesticides is ubiquitous, and over one million people are subjected to high occupational exposure. Pesticides and their residues are pervasive in our environment and our dependence upon pesticides is increasing. Although all pesticides sold in the US have passed Environmental Protection Agency (EPA) tests for carcinogenicity, exposure to pesticides have repeatedly been associated with increased human cancer incidence in epidemiologic studies. Our recent analysis of the Agricultural Health Study (AHS) cohort, the largest prospective study in the world of pesticide applicators from Iowa and North Carolina, also indicates that pesticide exposure increases risk for several cancers. The concern is that EPA's evaluation of chemical carcinogenicity is primarily based on the detection of mutagenic events. Altered DNA methylation, an emerging hallmark of cancer, is not considered in the EPA testing system. Emerging evidence suggests that promoter DNA methylation is affected by environmental exposures. Prior studies have shown that epigenetic information, including DNA methylation, contained in blood cell DNA represents a valuable predictive marker of cancer risk. Blood is a conduit between the external environment and human tissues, and as such constitutes a seminal target tissue to evaluate the effects of environmental toxicants on human health. Our preliminary data recently linked exposure to organophosphate pesticides (OPs), the most commonly used insecticides in the US, to increased promoter methylation of several tumor suppressor genes in blood DNA. We further confirmed this finding in vitro, using human blood cells treated with OPs. Taken together, we hypothesize that exposure to pesticides can induce promoter DNA methylation changes in blood cell DNA, and that this will provide currently unknown mechanistic insights into the association between pesticide exposure and cancer risk. We propose to study whether OP exposure alters gene promoter DNA methylation patterns in human subjects, and in OP-treated cell lines. We will first conduct a genome-wide screening in a subset of the population with the highest and lowest OP exposure, and in cell lines. Findings will then be validated using a gene-specific approach in an independent replication population, and after treatment of cells with varying doses of OP. We will also examine whether methyl donor-related nutrients and related genetic polymorphisms, two major factors in DNA methylation process, modify the associations between OP exposure and promoter DNA methylation levels. The investigations proposed in the current study will take advantage of the population and resources included in an existing NIEHS-funded cross-sectional study nested in AHS. PUBLIC HEALTH RELEVANCE: This study examines organophosphate pesticide (OP) exposure and neurological disorders in 700 AHS participants. Thus, we will have well-defined OP exposure data, DNA from blood samples, and comprehensive data for confounding and modifying effects that may impact data evaluation. Further, we have an established in-vitro system for evaluating epigenetic effects of pesticides. These are novel and innovative investigations, as DNA methylation studies with complementary human data and cell lines with pesticide treatment have not been done. This study will have high public health significance due to the widespread pesticide use in the US and worldwide. A major strength is the study population of farmers with well-characterized exposure assessment. Data on pesticide use, of this high quality and completeness, would be difficult to achieve in a general population cohort. Another major strength is that this study is guaranteed to be completed in a timely manner (if funded), since the DNA is ready for analysis, and other relevant data are also readily available. Pesticides are widely used in the US and worldwide, are pervasive in our environment. Our dependence upon pesticides is increasing. Although pesticides sold in the US have passed the Environmental Protection Agency (EPA) tests for their ability to cause cancer and been approved as safe, it has repeatedly been shown that some pesticides increased human cancer incidence. The concern is that EPA's evaluation on the safety of pesticides does not include the tests that examine DNA methylation alterations, a new emerging hallmark of cancer. Our proposed study is to investigate the effects of pesticides exposure on DNA methylation alterations in a subset of the population from the Agricultural Health Study (AHS), the largest prospective study in the world of pesticides applicators from Iowa and North Carolina. The results from this study will address a critical gap in our knowledge of pesticides and their ability to cause cancer.